The early diagnosis of certain diseases, especially cancer, can save many lives. In the case of cancer, and other diseases of genetic origin, early detection often depends on the availability of an appropriate analytical method which can accurately and reliably detect a putative mutation in DNA samples. This problem is exacerbated by the fact that such samples generally contain a very small population of cells containing mutant DNA in the presence of a very large predominantly normal cell population containing wild type DNA. Any separation technique which is capable of detecting mutant DNA in the presence of wild type would fail under these circumstances because the concentration of mutant DNA is simply too low to be detected relative to wild type. That is to say, the concentration of mutant DNA may be too low to detect in absolute terms. Alternatively, the concentration of mutant DNA may be sufficient to detect, but will be completely obscured because of the very large relative amount of wild type in the sample.
Increasing the amount of mutant DNA by PCR amplification of the sample would not solve the problem described above. The mutant and wild type DNA in the sample are very similar. In fact, their sequence may differ by only a single base pair. Therefore, the primers which would be used to amplify the mutant DNA would also amplify the wild type since both are present in the sample. As a result, the relative amounts of mutant and wild type DNA would not change.
Following radiation or chemotherapy, cancer patients are monitored for the presence of residual cancer cells to determine whether the patients are in remission. The effectiveness of these treatments can be monitored if small levels of residual cancer cells could be detected in a predominantly large wild type population. Traditionally, the remission status is assessed by a pathologist who conducts histological examination of tissues samples. However, these visual methods are largely qualitative, time-consuming, and costly. At best, the sensitivity of these methods permits detection of about 1 cancerous cell in 100 cells.
Analysis of DNA samples has historically been done using gel electrophoresis. Capillary electrophoresis has also been used to separate and analyze mixtures of DNA. However, these methods cannot distinguish point mutations from homoduplexes having the same base pair length.
Gel based analytical methods, such as denaturing gradient gel electrophoresis and denaturing gradient gel capillary electrophoresis, can detect mutations in heteroduplex DNA strands under "partially denaturing" conditions. The term "partially denaturing" means the separation of a mismatched base pair (caused by temperature, pH, solvent, or other known factors) in a DNA double strand while the remainder of the double strand remains intact. However, these gel based techniques are operationally difficult to implement and require highly skilled personnel. In addition, the analyses are lengthy and require a great deal of set-up time. A denaturing capillary gel electrophoresis analysis of a 90 base pair fragment takes more than 30 minutes and a denaturing gel electrophoresis analysis may take 5 hours or more. The long analysis time of the gel methodology is further exacerbated by the fact that the movement of DNA fragments in a gel is inversely proportional, in a geometric relationship, to their length. Therefore, the analysis time of longer DNA fragments can be often be untenable. Sample recovery of DNA fragments separated on a gel is difficult and time consuming, requiring specialized techniques.
In addition to the deficiencies of denaturing gel methods mentioned above, these techniques are not always reproducible or accurate since the preparation of a gel slab and running an analysis can be highly variable from one operator to another. As a result, the mobility of a DNA fragment is often different on different gel slabs and even in one lane, compared to another on the same gel slab. The problems and deficiencies of gel based DNA separation methods are well known in the art and are described in "Laboratory Methods for the Detection of Mutations and Polymorphisms", ed. G. R. Taylor, CRC Press (1997).